Thermal imaging during photodynamic therapy (PDT)

This study was designed to measure the PDT associated changes in tumour tissue temperature and to calculate if tumoricidally relevant hyperthermia might be induced by the current clinically used PDT modalities. Syrian golden hamsters with amelanotic melanomas (A-Mel-3) received intravenous injections of Photofrin II (5 mg kg⁻¹ b.w.) or 0.9% sodium chloride (2 ml kg⁻¹ b.w.). Twenty-four hours later tumour surface and tumour centre temperature were quantified by an infra-red sensitive thermal imaging device and an implanted thermal probe during laser treatment at a wavelength of 630 nm. With laser light irradiation using power densities of 100 mW cm⁻² or 200 mW cm⁻², a significant temperature increase was noted in comparison to the baseline values. At total energy delivery of 100 J cm⁻² the tumoricidally relevant threshold of 43°C was exceeded with 200 mW cm⁻² only. However, no significant temperature differences were measured between photosensitized and control tumours receiving 100 mW cm⁻² or 200 mW cm⁻². Calculation of the corresponding applied hyperthermic dose revealed that submaximum hyperthermic effects were involved with irradiation intensities of 200 mW cm⁻².     

Evaluation of the Third Generation Photosensitiser SC102 in Two Animal Models

. SC102, a poly(ethylene glycol) (PEG-2000) derivative of the second generation photosensitiser temoporfin (Foscan), was evaluated for photodynamic activity utilising two standard animal models: the rabbit, inoculated with the cottontail rabbit papilloma virus (CRPV) and the healthy canine larynx. Optimal drug dose and drug–light interval (DLI) were determined in a series of pharmacokinetic experiments using SC102 administered intravenously at doses of 3 and 30 mg/kg in our standard CRPV rabbit model. Plasma pharmacokinetics in the rabbit showed an elimination half-life of 121±20 h. Peak tumour concentration occurred at 144 h for the 3 mg/kg group and at 240 h for the 30 mg/kg group. The disposition ratio of SC102 between tumour to healthy tissue, at peak tumour levels, was approximately 4 to 1 for both dose levels. Skin tolerance to increasing 652 nm wavelength fluences was excellent. Even in those rabbits given a 30 mg/kg SC102 dose, no significant damage to the skin was observed, even when a fluence of 160 J/cm² was applied at the optimal drug–light interval of 10 days. Tumour efficacy of SC102 PDT was evaluated in rabbits previously inoculated with CRPV. One time photoactivation of a single 30 mg/kg dose of SC102 at a DLI of 10 days using a fluence of 100 J/cm² achieved a complete tumour clearance rate of 35%. Two-time photoactivation on days 6 and 10 after single administration of the same dose in a separate group of rabbits, using a fluence of 75 J/cm² on both occasions, yielded an improved cure rate of 58%. Evaluation of normal tissue tolerance to SC102 PDT was also investigated in the healthy dog larynx model 10 days after an intravenous dose of 30 mg/kg SC102. A fluence of 200 J was determined to be the maximum tolerated dose at which time there was a 4:1 ratio of SC102 between laryngeal mucosa and muscle. The photophysical and pharmacokinetic profiles of SC102 show significant differences from those of Foscan from which it is derived. In these preliminary in vivo experiments we have demonstrated the outstanding tolerance of normal tissues to SC102 PDT using high fluences. We have also shown tumour efficacy with light doses readily achievable in the clinical setting. Disposition of SC102 in the skin is low and photosensitivity risk may disappear within the drug–light interval. Based on these conclusions we believe SC102 PDT may have potential utility as an adjunct to surgical resection of tumours necessitating wide field exposure of normal tissue to activating light. Paper received 15 January 1999; accepted after revision 6 October 1999.     

Use of photodynamic therapy in the treatment of oral and maxillofacial cancer and precancer: Report of 104 cases and preliminary observation on the mechanism

This paper describes the use of photodynamic therapy (PDT) in the treatment of 104 patients with premalignant or malignant disease of the oral cavity and maxillofacial regions. The tumours in 10 patients were submitted to detailed histological examination following resection 24 hours after PDT. In all patients the photosensitizer was haematoporphyrin derivative given in a dose of 2.5–5.0 mg/kg body weight, 48–72h before photoirradiation. Einghteen patients were treated with the helium-neon laser, with a power density of 120–200 mW/cm² and a total energy dose of 200–500 J/cm², but in this group a complete response was obtained in only 39% of patients. However, when the argon-pumped dye laser was used in the remaining cases, with a power density of 400–800 mW/cm² and a total energy dose of 720–1440 J/cm², the complete response rate rose to 72%. In the latter group, 15 patients were treated by fibre-optic implantation between the tumour and underlying normal tissues. From the histological study it would appear that PDT acts both on the blood vessels of the stroma and also at a cellular level, with some possible enhancement of immune function. Indications for PDT, based on this study, are defined.     

Safety studies for intraoperative photodynamic therapy

A new adjunctive therapy is needed for colorectal carcinoma surgery to decrease local recurrence rates. Photodynamic therapy (PDT) may be able to fulfil this role by activating the selective photosensitizer haematoporphyrin derivative (HPD) intraoperatively with laser light. This technique would necessitate the irradiation of normal tissues and therefore safety studies have been carried out in miniature pigs. Animals were photosensitized with HPD (5 mg kg⁻¹), then 48 h later colonic anastomoses and ureters were irradiated with 50 J cm⁻² of 510 nm-equivalent light. Anatomical, physiological and biochemical analyses were carried out, investigating both structure and function. Our results show that PDT applied in a potentially useful biological dose has no detrimental effect on the healing of anastomoses and ureteric structure and function. This work paves the way for intraoperative adjunctive PDT to be used effectively in man.     

Optimisation of Illumination for Photodynamic Therapy With mTHPC on Normal Colon and a Transplantable Tumour in Rats

. Recent reports suggest that the effect of photodynamic therapy (PDT) can be enhanced by fractionating the light dose or reducing the light fluence rate. We assessed these options on two tissues in rats (normal colon and a transplanted fibrosarcoma) using the photosensitiser meta-tetrahydroxyphenylchlorin (mTHPC). Animals were sensitised with 0.3 mg/kg mTHPC, 3 days prior to illumination with red light (652 nm) using a single fibre touching the target tissue and killed 1–3 days later for quantitative measurement of the extent of PDT necrosis. Results were similar for both tissues, although the differences between illumination regimens were less marked in tumour tissue. Using continuous illumination and a fixed low energy in colon, the extent of necrosis was up to almost three times larger with 5 mW than with 100 mW, although the maximum attainable necrosis was independent of power. The long treatment time using 5 mW could be halved without loss of effect by increasing the power during treatment. Dividing the light into two equal fractions at 100 mW increased the lesion size by up to 20% in colon (independent of the timing of the dark interval), but by only 10% in tumour and had no effect at 20 mW. Previous studies using 5-aminolaevulinic acid (ALA) showed a much larger effect of fractionation that was critically dependent on the timing of the dark interval. We postulate that enhancement of PDT by fractionation is due to improved oxygen supply to the treated area which may be due to reversal of temporary vascular occlusion (more likely with ALA) or less rapid photochemical consumption of oxygen (more likely with mTHPC). At lower fluence rates, the oxygen consumption rate is not fast enough to be improved by fractionation. We conclude that fractionated or low power light delivery can enhance PDT with mTHPC. Although the effects are not large, this may be of value for interstitial treatment of solid tumours when multiple sites are treated simultaneously. Paper received 9 April 2001; accepted after revision 28 September 2001.     

Development of an alternative light source to lasers for photodynamic therapy: 1. Comparative in vitro dose response characteristics

The relative performances of a prototype lamp, a pulsed laser and a continous wave laser, were compared for photodynamic therapy (PDT). Recent advances in short are technology and lamp miniaturization coupled with improvements in the effciency, of optical filter coatings have led to the design and construction of a table-top light source prototype; the first viable and cost-effective alternative to a laser, particularly in the field of PDT. The device can deliver over 1 W directly or 0.5W via a light guide within a 30 nm band centred at any wavelength from the ultra-violet to the near infra-red at fluence rates of over 1 W cm⁻², in excess of that required for PDT. Its relative biological effectiveness (RBE), in vitro, has been proven alongside two PDT laser systems, an argon pumped dye laser and a copper vapour pumped dye laser. These first in vitro tests showed an efficiency of haematoporphyrin derivative, (HPD) induced cellular photoinactivation close to that of the argon/dye laser (RBE 100%), with a mean RBE for the lamp of 87±3% (p<0.05). The lamp proved to be superior, to that of the copper/dye laser system with an RBE of up to 150% at fluence rates above 50 m W cm⁻². Transient photobleaching of the photosensitizer was the probable cause for the relative ineffectiveness of the copper/dye laser for PDT at high fluence rates.     

Photodynamic therapy of tumours and other diseases using porphyrins

Photodynamic therapy (PDT) with porphyrins and red light (620–630 nm) is finding increasing clinical application for both the eradication of relatively small tumours and the palliation of inoperable or obstructive tumours. PDT also shows some promise for the sterilization of the tumour bed after surgical removal of neoplastic masses. Several porphyrins have been found to be accumulated and retained by tumour tissues; however, a chemically prepared derivative of haematoporphyrin, termed HpD, and a purified form of HpD, termed DHE (dihaematoporphyrin ether or ester), are most frequently used in clinical practice owing to their optimal tumour-localizing properties and low systemic toxicity in the dark. The efficiency of HpD/DHE photoactivation by red light is very low, since their extinction coefficient at wavelengths above 600 nm is below 10³ m ⁻¹ cm⁻¹. Therefore, a large number of investigations are being performed in order to improve the efficacy of PDT. One approach involves the use of porphyrin analogs (e.g., chlorins, phthalocyanines) which retain a high affinity for tumours and possess intense absorption bands in the red spectral region. Moreover, the selectivity of tumour targeting can be enhanced by transport of the photosensitizing drug with some types of lipoproteins or monoclonal antibodies. These developments are of interest also in view of the proposed extension of PDT to the treatment of other diseases, including viral and microbial infections, atheroma and psoriasis.     

Photodynamic therapy of experimental tumours with Zn(II)-phthalocyanine and pulsed laser irradiation

The effectiveness of a pulsed dye laser (673 nm) for photodynamic therapy (PDT) of tumours in the presence of Zn(II)-phthalocyanine (ZnPc) was evaluated using Lewis lung carcinoma-bearing mice. The tumours were irradiated with different pulse energies (from 0.4 to 10 mJ) at a constant fluence of 0.6 J cm^–2 at 24 h after administration of 0.25 mg kg^–1 body weight liposome-incorporated ZnPc. Maximal PDT effect, as evaluated by changes in mean tumour diameter, animal survival time and histological evaluation of tumour necrosis, was observed after 3.0 mJ pulse energy irradiation which appears to yield a deeper light penetration and a more efficient sensitizer excitation when compared with lower or higher pulse energies. Electron microscopic analysis of photo-treated tumour indicates preferential damage to malignant tissue as compared to endothelial cells.     

Development of an alternative light source to lasers for photodynamic therapy: 2. Comparative in vivo tumour response characteristics

The performance of a low cost, table-top/portable light source was tested against an argon ion pumped dye laser for in vivo photodynamic therapy (PDT). The prototype delivers up to 1 W via a 4 mm flexible lightguide within a 30 nm bandwidth centred at any wavelength from 300 nm to 1200 nm at fluence rates of up to 8 W cm^–2. An in situ bioassay using regrowth delay of tumour T50/80 was used to quantify the relative efficacy of the prototype with a laser. The tumours were sensitized with haematoporphyrin derivative (HpD) and externally irradiated. There was no significant difference in the response of the tumour to treatment between the two light sources (p = 0.69). Mean growth delays ranged from 2 days (light dose 10 J cm^–2) to 20 days (light dose 100 J cm^–2). The estimate for the difference in means (laser minus prototype growth delay) was only 0.66 days and was not statistically significant. This in vivo study demonstrates that the prototype is equivalent to a laser in PDT effect. The device has low capital/running cost, is simple to use and is one of the most powerful, spectrally efficient non-laser PDT sources available.     

Photodynamic therapy for the treatment of advanced gastrointestinal tumours

In the study, 120 patients with advanced gastrointestinal tumours were treated by PDT; 5 mg/kg of HpD was intravenously given 48–72 h prior to PDT. The light source was an argon dye laser with an output beam of 630 nm. The irradiation time varied from 15–25 min with a power of 100–350 mW cm^–2. The entire tumour was irradiated with a light dose of 100–250 J cm^–2. Of the 120 patients, 20 had cancer of esophagus, 72 had cancer of the gastric cardia, 18 had cancer of the stomach and 10 had cancer of the rectum. Eighty-eight patients (73.3%) had a response to PDT. Twelve patients with CR were followed up for one to five years, two patients died during the two years after PDT.     

Misonidazole combined with photodynamic therapy of rat 9L gliosarcoma tumours

Fifty-six Fischer 344 rats bearing subcutaneous 9L-gliosarcoma tumours were studied to determine if Misonidazole (MISO), combined with photodynamic therapy (PDT), would be more effective than PDT alone. PDT, like conventional radiation, is potentiated by oxygen, and if there are areas of hypoxic cells within the tumour it is possible that the addition of the radiosensitizing drug should make the treatment more effective. Thirty-nine rats were divided into eight groups as controls. Seventeen rats were divided into three groups and received MISO combined with PDT, five rats were exposed to a laser dose of (nm = 630) 300 J at 300 mW, seven rats to a laser dose of 600 J at 600 mW and five rats to a laser dose of 2160 J at 600 mW. The tumours were approximately 1 cm³ when treated. Animals treated with either PDT at 300 J or 600 J failed to show any effect on growth of the tumour. At 2160 J a definite delay in growth was observed but addition of MISO did not potentiate this effect. The results indicate this combined therapy did not slow the growth rate of the tumours in this model. The implication of these results are discussed.     

Photodynamic therapy to the oral cavity,tongue and larynx: a canine normal tissue tolerance study

Photodynamic therapy (PDT) has the potential to treat early carcinomas of the oral cavity and larynx while preserving normal tissue. However, normal tissues retain the photosensitizing agents and may be activated by high light fluence and dose rates resulting in normal tissue necrosis. The effects of varying dose rates of light delivery on various tissues in the upper aerodigestive tract have not been evaluated to date and are necessary to determine a therapeutic light dose range that will result in selective tumour necrosis. Thirty adult mongrel dogs received intravenous Photofrin, 2 mg kg^–1, 48 h prior to PDT treatment. Photodynamic therapy was administered to the tongue, buccal mucosa and larynx with a microlens fibre and implantable cylindrical diffuser at various dose rates from 20 to 125 J cm^–2 at 150 mW cm^–2. At the same dose rate of light delivery, the tongue was the most sensitive organ, followed by the buccal mucosa, and last by the larynx. The differential tissue effect of identical dose rates of therapy must be taken into account when administering PDT so that selective tumour necrosis with normal tissue preservation may be achieved. This study indicates the need to perform evaluations of the effect of PDT on other tissue types in an animal model with each new photosensitizer prior to administering PDT to those areas in humans.     

Application of lower fluence rate for less microvasculature damage and greater cell-killing during photodynamic therapy

During the process of photodynamic therapy (PDT), problems arise such as stasis or occlusion of microvasculature, tumor oxygen depletion, and photosensitizer bleaching. This study shows that the first problem could be reduced by using a lower fluence rate light source in PDT. Microvasculature damage was studied experimentally in hematoporphyrin derivative–mediated PDT against light fluence rate, and, to some extent, less microvasculature damage was induced under 75 mW/cm² illumination than under 150 mW/cm². Histology of vessels at the end of PDT showed that vessel damage could be observed in both groups, indicating that the microvasculature would eventually be damaged as long as the administration of light fluence was sufficient and regardless of the illuminating fluence rates. Thus microvasculature damage induced by low fluence rate illumination could also be effective in tumor control after PDT. The cell-killing experiment was performed in vitro and designed so that cell-killing rate was influenced only by light characteristics. The higher cell-killing rate caused by 75 mW/cm² illumination indicated that lower fluence rate light could enhance the light absorbency or decrease the bleaching of photosensitizer.An erratum to this article can be found at     

Application of lower fluence rate for less microvasculature damage and greater cell-killing during photodynamic therapy

During the process of photodynamic therapy (PDT), problems arise such as stasis or occlusion of microvasculature, tumor oxygen depletion, and photosensitizer bleaching. This study shows that the first problem could be reduced by using a lower fluence rate light source in PDT. Microvasculature damage was studied experimentally in hematoporphyrin derivative–mediated PDT against light fluence rate, and, to some extent, less microvasculature damage was induced under 75 mW/cm² illumination than under 150 mW/cm². Histology of vessels at the end of PDT showed that vessel damage could be observed in both groups, indicating that the microvasculature would eventually be damaged as long as the administration of light fluence was sufficient and regardless of the illuminating fluence rates. Thus microvasculature damage induced by low fluence rate illumination could also be effective in tumor control after PDT. The cell-killing experiment was performed in vitro and designed so that cell-killing rate was influenced only by light characteristics. The higher cell-killing rate caused by 75 mW/cm² illumination indicated that lower fluence rate light could enhance the light absorbency or decrease the bleaching of photosensitizer.The online version of the original article can be found at     

Photodynamic therapy: Optimal treatment parameters in murine fibrosarcoma

We have investigated the efficacy of photodynamic therapy (PDT) by using two preparations of haematoporphyrin derivative (HPD) that demonstrated different biological activities against experimental murine fibrosarcoma RIF-1 in C3H/He mice. We have been able to demonstrate a minimum clonogenic survival rate of 0.25% by using the more active HPD at 20 mg/kg with a 40-h retention time and a total laser light dose of 100 J/cm². Further, we noted that clonogenic survival rates of 21.6% and 25.0% respectively could be achieved by using the less active HPD (at 20 mg/kg) with a laser light dose of 150 J/cm², or the more active HPD (at 20 mg/kg) with a laser light dose of 20 J/cm². In both cases necrosis of the surrounding normal tissue was absent. Necrosis of the normal tissue surrounding the tumour was shown to be associated with high laser energy (100 J/cm² and higher) in conjunction with a high dose (20 mg/kg) of the more active HPD. A comparison of survival curves as a function of laser energy for the RIF-1 cells following PDT with the two different preparations of HPD showed a difference in the kinetics of cell death. A curve with a shoulder region and aD _o (mean lethal dose) of 41 J/cm² was obtained when the less active HPD was used, whereas PDT using the more active HPD resulted in a curve with no shoulder and aD _o of 16 J/cm².     

Aminolaevulinic Acid-induced Photodynamic Therapy in the Treatment of Dysplastic Barrett's Oesophagus and Adenocarcinoma

. Photodynamic therapy (PDT) may have a role in the prevention of oesophageal cancer. Ten patients with Barrett's oesophagus, three with low-grade dysplasia (LGD), four with high-grade dysplasia (HGD), one with carcinoma in situ and two with invasive carcinoma, were treated with PDT. All received 30 mg/kg aminolaevulinic acid (ALA) followed 4 h later by laser endoscopy. Half were treated with red light (630 nm; 100 mW/cm² for 1000 s) and half with green light (514 nm; 100 mW/cm² for 500 s). Columnar epithelial regression was seen in all patients with dysplasia (mean area decrease 44%; range 10–100%), with apparent elimination of dysplasia in all cases. In patients with in situ or invasive carcinoma, no response was seen. ALA-induced PDT, using either red or green light, produces effective ablation of dysplastic Barrett's oesophagus, hence may have a role in the prevention of oesophageal carcinoma, but has little effect on in situ or invasive adenocarcinoma. Paper received 3 January 1999; accepted after revision 12 April 1999.     

Near infra-red diode laser for photodynamic tumour therapy using bacteriochlorina

Clonogenic cell survivals were performed in order to assess the feasibility of tumour cell kill with an experimental diode laser emitting 250 mW of light at λ = 779 nm using the photosensitizer bacteriochlorina(BCA). The AlGaAs diode laser is based on organometallic vapour epitaxial crystal growth technology. The electrical to optical conversion efficiency amounts to 21% and the beam divergence is 47° by 7.0° full width at half maximum. BCA was proved to be an effective non-toxic photosensitizer in vitro and in vivo. It has a major absorption peak at 760 nm where tissue penetration of light is optimal. Clonogenic T24 human bladder carcinoma cell survivals were photosensitizer concentration and light dose dependent. A 0.1% survival rate was obtained with an illumination intensity of 50 mWcm⁻² for 90 s (4.5 Jcm⁻²) and a BCA concentration of 6 μgml⁻¹. Illumination without BCA at energy levels exceeding the PDT levels with a factor 10, or BCA alone without illumination had no effect on the cells in the clonogenic cell survivals. The combination of BCA with a near infra-red diode laser is most promising for photodynamic tumour therapy as a result of the reliability, compactness and relatively low price of the illumination device, the high transmittance of near infra-red light in tissue and the tumour killing potential of BCA.     

Development of a novel indwelling balloon applicator for optimizing light delivery in photodynamic therapy

BACKGROUND AND OBJECTIVE: A human glioma spheroid model is used to investigate the efficacy of different light delivery schemes in 5-aminolevulinic acid (ALA)--mediated photodynamic therapy (PDT). The results provide the rationale for the development of an indwelling balloon applicator for optimizing light delivery. STUDY DESIGN/MATERIALS AND METHODS: Human glioma spheroids were incubated in ALA (100 or 1000 microg /ml-1) for 4 hours and subjected to various light irradiation schemes. In one set of experiments, spheroid survival was monitored as a function of light fluence rate (5-200 mW cm-2). In all cases, spheroids were exposed to fluences of either 25 or 50 J cm-2. In a second study, the effects of repeated weekly PDT treatments, using sub-threshold fluences, were investigated. One group of spheroids was subjected to three treatments using fluences of 12, 12, and 25 J cm-2. Results were compared to spheroids receiving single treatments of either 12 or 25 J cm-2. A fluence rate of 25 mW cm-2 was used for all three groups of spheroids. In all cases, the effect of a given irradiation scheme was evaluated by monitoring spheroid growth. RESULTS: Low fluence rates produce greater cell kill than high fluence rates. The minimum effective fluence rate in human glioma spheroids is approximately 10 mW cm-2. Repeated weekly PDT treatments with sub-threshold fluences result in significant cell kill. In spheroids surviving the PDT treatments, growth is suppressed for the duration of the treatment period. CONCLUSION: The results of the in vitro studies support the development of an indwelling balloon applicator for the delivery of light doses in long term multi-fractionated PDT regimens.     

Effect of Helium-Neon (He-Ne) Laser Irradiation on Dog Neoplasm Cells in Culture

The effects of laser light on the cellular proliferation have been extensively characterised. Low-power laser sources, such as the helium–neon (He-Ne) laser irradiation with a wavelength of 632.8 nm, have been found to produce photobiological and photodamaging effects with evidence of interference with cell proliferation functions. The present study has investigated the in vitro effect of He-Ne laser irradiation on the proliferative action of dog tumour cells in culture. Dose–response studies showed that repeated He-Ne irradiation (irradiance 12.8 mW/cm²) once a day for 4 consecutive days in a dose range between 0.13 and 2.08 J/cm² significantly increased with increasing energy density up to a laser dose of 0.26 J/cm², whereas at >1.04 J/cm², the cell proliferation decreased with increasing energy densities. It is concluded that the application of He-Ne laser irradiation at energy densities ranging from 0.13 J/cm² to 2.08 J/cm² produced different effects on cell proliferation in dog tumour cells in culture. Paper received for publication 27 June 1997; accepted following revision 6 February 1998.     

Interstitial photodynamic therapy in the Dunning R3327-AT6 prostatic carcinoma

Interstitial photodynamic therapy (PDT) could be an alternative radical treatment for prostate cancer. The ability to predict the depth of necrosis is necessary for light treatment planning using multiple optical fibres. The extent of PDT necrosis was studied in subcutaneously implanted R3327-AT6 Dunning prostate tumours which had similar optical characteristics to human prostate. Tumour-bearing subjects were given 20 mg kg^–1 Haematoporphyrin esters (HPE) and irradiated 24 h later with 630 nm laser light. Five subjects per group were treated with increasing light doses (50–450 J cm^–1) delivered interstitially via a single 2 cm long cylindrical diffuser. After 450 J cm^–1 of irradiation, 4.3±0.8 cm³ [standard error of the mean (s.e.m.)] of tumour tissue was necrosed to a depth of 10.5±0.8 mm around the diffuser. There was an approximately linear correlation between the volume of PDT necrosis around the fibre and prescribed light dose. The mean threshold light dose for PDT effect was 18±2 J cm^–2. In this tumour with a mean photosensitizer concentration of 16±1.5g g^–1, low light doses produced tumour necrosis. PDT using multiple diffusers could destroy a relatively large tumour volume and the diffusion theory model reliably predicted the depth of necrosis.